CN102212620A - Measurement and audit kit for detecting three fish rhabdoviruses - Google Patents
Measurement and audit kit for detecting three fish rhabdoviruses Download PDFInfo
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Abstract
The invention discloses a measurement and audit kit for detecting three fish rhabdoviruses. The kit contains specific nucleic acid samples of the three fish rhabdoviruses, can simultaneously provide a group of randomly combined measurement and audit samples for the distinguishing detection of nucleic acids of the three viruses including IHNV (infectious hematopoietic necrosis virus), VHSV (viral hemorrhagic septicaemia virus) and SVCV (spring viraemia of carp virus), and provides an evaluation benchmark tool for the sensitivity and specificity of participating mechanisms in the testing detection of the nucleic acids of the three viruses as well as the accuracy of the detection method by means of qualitative evaluation.
Description
Technical field
The present invention relates to the measurement audit test kit of three kinds of kinds of fish Rhabdovirus of a kind of detection.
Background information
Infectivity hematopoietic tissue necrosis virus (Infectious hematopoietic necrosis virus, IHNV), viral haemorrhagic septicaemia virus (Viral hemorrhagic septicemia virus, VHSV) and SVCV (Spring Viraemia of Carp Virus, SVCV) being three kinds of fish viruses that belong to Rhabdoviridae (Rhabdoviridae) together, also is three kinds of important viruses that influence culture fishery.Wherein, IHNV and VHSV belong to the grain an outer Rhabdovirus (
Novirhabdovirus), SVCV belong to Vesiculovirus belong to (
Vesiculovirus), they all are the negative burst RNA viruses of strand.These three kinds of viruses are distributed widely in many areas in Europe, America and Asia, can infect multiple fish, especially to fry and juvenile fish, can cause higher mortality ratio, all can reach more than 90% when serious.Infectious hematopoietic necrosis (the Infectious hematopoietic necrosis that causes by these three kinds of viruses respectively, IHN), viral hemorrhagic septicemia (Viral hemorrhagic septicemia, VHS) and spring viremia (Spring Viraemia of Carp, SVC) in case outburst, will give the loss of bringing on a disaster property of aquatic products aquaculture, so their three kinds of fish eqpidemic diseases that to be countries in the world actively prevent makes.
At present, the domestic regional testing laboratory that many coastal laboratories and the prosperity of landlocked aquatic products aquaculture industry arranged all carries out the detection about these three kinds of viruses.Method for detecting virus commonly used is cell cultures isolated viral method, serological method and polymerase chain reaction (PCR).Cell cultures isolated viral method reliable results, but sense cycle is long, is not suitable for rapid differential diagnosis, and need additive method to carry out follow-up detection toward contact, could determine viral species.Common RT-PCR and real-time fluorescence RT-PCR The Application of Technology have improved the specificity and the sensitivity that detect, have shortened detection time, have obtained certain effect.Williams etc. (1999) have also reported for work and have used multiple RT-PCR to detect the research of IPNV, IHNV and VHSV, the working efficiency when having improved multiple virus greatly and detecting.
Summary of the invention
The technical problem to be solved in the present invention provides the measurement audit test kit of three kinds of kinds of fish Rhabdovirus of a kind of detection.This test kit mainly uses in the measurement audit field of Animal Quarantine specialty, is used for the test kit that three kinds of kinds of fish Rhabdovirus abilities are detected in the control experiment chamber, and promptly this test kit can be used for estimating the examination laboratory and whether can correctly detect three kinds of kinds of fish Rhabdovirus.
For achieving the above object, the present invention is by the following technical solutions:
Test kit is used in the measurement audit of three kinds of kinds of fish Rhabdovirus of a kind of detection, is stored in-20 ℃, and this test kit is made up of following sample:
A: contain 10
7The solution of copy/mL purpose nucleic acid fragment, described purpose nucleic acid fragment are the RNA fragment of viral haemorrhagic septicaemia virus (VHSV) in-vitro transcription, and its corresponding DNA sequences is the nucleotide sequence shown in the sequence table SEQ ID No.1;
B: contain 10
8The solution of copy/mL purpose nucleic acid fragment, described purpose nucleic acid fragment are the RNA fragment of viral haemorrhagic septicaemia virus (VHSV) in-vitro transcription, and its corresponding DNA sequences is the nucleotide sequence shown in the sequence table SEQ ID No.1;
C: contain 10
7The solution of copy/mL purpose nucleic acid fragment, described purpose nucleic acid fragment are the RNA fragment of the sick virus of infectivity hematopoietic tissue necrosis (IHNV) in-vitro transcription, and its corresponding DNA sequences is the nucleotide sequence shown in the sequence table SEQ ID No.2;
D: contain 10
8The solution of copy/mL purpose nucleic acid fragment, described purpose nucleic acid fragment are the RNA fragment of the sick virus of infectivity hematopoietic tissue necrosis (IHNV) in-vitro transcription, and its corresponding DNA sequences is the nucleotide sequence shown in the sequence table SEQ ID No.2;
E: contain 10
7The solution of copy/mL purpose nucleic acid fragment, described purpose nucleic acid fragment are the RNA fragment of SVCV (SVCV) in-vitro transcription, and its corresponding DNA sequences is the nucleotide sequence shown in the sequence table SEQ ID No.3;
F: contain 10
8The solution of copy/mL purpose nucleic acid fragment, described purpose nucleic acid fragment are the RNA fragment of SVCV (SVCV) in-vitro transcription, and its corresponding DNA sequences is the nucleotide sequence shown in the sequence table SEQ ID No.3;
G: the solution that does not contain the purpose nucleic acid fragment.
Wherein, described solution is preferably 75% ethanolic soln.
The measurement audit of the present invention using method of test kit:
1. the world, country and industry standard method or other nonstandard methods can be selected in each laboratory of participating in the experiment, and carry out the detection validation of three kinds of kinds of fish Rhabdovirus nucleic acid;
2. test kit specification sheets
2.1 storage temperature :-20 ℃;
2.2 validity period: 6 months;
2.3 using method: put upside down the mixing liquid in pipe before the use, and then extracting RNA nucleic acid carries out test analysis;
2.4 cycling condition setting:
Fs, 42 ℃ of 30 min of reverse transcription;
Subordinate phase, pre-92 ℃ of 3 min of sex change;
Phase III, 92 ℃ of 10 s; 45 ℃ of 30 s; 72 ℃ of 1 min; 5 circulations;
The quadravalence section, 92 ℃ of 10 s; 60 ℃ of 30 s; Fluorescence is collected in 40 circulations when the each round-robin annealing of quadravalence section is extended;
After test detects and finishes, according to the fluorescence curve and the Ct value result of determination of collecting;
2.5 relative reference Ct value: be as the criterion 10 with the loop parameter in 2.4
7The relative reference Ct value of copy/mL purpose nucleic acid fragment solution is about 17,10
8The relative reference Ct value of copy/mL purpose nucleic acid fragment solution is about 13.
3. evaluation of result
If do not detect the specific RNA sequence in this test kit, illustrate that then this laboratory detection technology of participating in the experiment has problem; If can detect the specific purpose nucleic acid fragment in this test kit, and the Ct value that detects and the Ct value employing two sample mean test of significance-t that demarcates check and carry out statistical study and do not have significant difference, proves that then this laboratory detection technology level of participating in the experiment is good; If can detect the specific purpose nucleic acid fragment in this test kit, two sample mean test of significance-statistical study is carried out in the t check, and there were significant differences but the Ct value that detects adopts with the Ct value of demarcating, then prove this not enough standard of laboratory detection technology level and stable of participating in the experiment, need to improve.
Novelty of the present invention: this test kit can be simultaneously for the detection of differentiating IHNV, VHSV and three kinds of viral nucleic acids of SVCV provides one group of measurement audit sample by combination at random, the accuracy of susceptibility, specificity and the detection method that these three kinds of viral nucleic acids of test of the mechanism that participates in the experiment is detected with the mode of qualitative evaluation provides the benchmark instrument of testing and assessing.This test kit has fine reference value for the evaluation mechanism that participates in the experiment to the detectivity of three kinds of kinds of fish Rhabdovirus nucleic acid, and the detectivity that promotes associated mechanisms is had very strong promoter action.
Three kinds of viral nucleic acids in this test kit are prepared according to the requirement of reference material; adopted cooperation to demarcate, calculate uncertainty, homogeneity and stability test etc.; modulated and be applicable to the best sample concentration of measuring audit; proof has adopted 75% ethanolic soln the highest as cost performance by experiment, can effectively protect and disperse the dispersion protective material of RNA nucleic acid again.The preparation that the Animal Quarantine viral nucleic acid is measured the audit sample has been proposed first, and it is quantitative.
The invention will be further described below in conjunction with specification drawings and specific embodiments, all any this areas of doing according to the disclosure of invention be equal to replacement, all belong to protection scope of the present invention.
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.
The used viral haemorrhagic septicaemia virus of the present invention (Viral hemorrhagic septicemia virus, VHSV), the sick virus of infectivity hematopoietic tissue necrosis (Infectious hematopoietic necrosis virus, IHNV), SVCV (Spring viraemia of carp virus, SVCV) and EPC clone (Epithelioma papulosum cyprini) preserve by Inspection and Quarantine Technology Center, Beijing Entry-Exit Inspection and Q Animal Lab..
Material source used in the present invention: cloning vector pGEM-T Easy Vector, available from (the total length 3015bp of promega company, carry ampicillin resistance gene (Amp) and Lac gene, recombinant chou can be by blue hickie and the dual Screening and Identification of ammonia benzyl resistance); E.coli DH5 α competent cell is available from TIANGEN Biotech (Beijing) Co., Ltd.; TRIZOL Reagent is available from Invitrogen company; Taq archaeal dna polymerase, dNTP, M-MLV ThermoScript II (M-MLV Reverse Transcriptase), recombinant RNA enzyme inhibitors (Recombinant RNasin Ribonuclease Inhibitor) are all available from promega company; TakaRa DNA Fragment Purification Kit Ver 2.0; TaKaRa MiniBEST Plasmid Purification Kit Ver 2.0 is all available from precious (Dalian) biotechnology company limited; Promega RiboMAX Large Scale RNA Production System-T7 is available from Progema company; Ready-To-Go RT-PCR Beads is available from Amersham Biosciences company.
The preparation that 1: three kind of kinds of fish Rhabdovirus of embodiment is measured the audit sample
1. be designed for the primer of three kinds of kinds of fish Rhabdovirus N genes of amplification respectively
(1) viral haemorrhagic septicaemia virus (VHSV) primer design is with synthetic
Complete genome sequence (sequence number: AF143862, AF143863, X73873, Z93412, Z93414) according to the VHSV that logins among the GenBank, compare with DNAMAN software, conservative region is selected at two ends at virus N-gene, use Oligo 6.0 software designs to go out a pair of primer (VP1 and VP2), this primer can amplify the fragment of 1278 bp, and sequence is as follows:
VP1：5’-ATGGAAGGAGGAATTCGTGCAG-3’?（SEQ?ID?No.4）
VP2：5’-GAGAAATTCTTATAATCGTGCCG-3’?（SEQ?ID?No.5）
(2) infectious hematopoietic necrosis (IHNV) primer design is with synthetic
Complete genome sequence (sequence number: L40883, NC001652, X89213) according to the IHNV that logins among the GenBank, compare with DNAMAN software, at the N of virus gene Selection conservative region, use Oligo 6.0 software designs to go out a pair of primer (IP1 and IP2), this primer can amplify the fragment of 977 bp, and sequence is as follows:
IP1：5’-TCACGAACGATGACAAGCGCACTC-3’?（SEQ?ID?No.6）
IP2：5’-?ACCTTTATCCCTGTCAGTCCTCTC-3’?（SEQ?ID?No.7）
(3) SVCV (SVCV) primer design is with synthetic
Complete genome sequence (sequence number: AJ318079, DQ097384, DQ491000, NC002803, U18101) according to the SVCV that logins among the GenBank, compare with DNAMAN software, conservative region is selected at two ends at virus N-gene, use Oligo 6.0 software designs to go out a pair of primer (SP1 and SP2), this primer can amplify the fragment of 1713 bp, and sequence is as follows:
SP1：5’-GTTCCTTCCCTGTTCATGGGAG-3’?（SEQ?ID?No.8）
SP2：5’-AATCCAGACGGAGTATCTTGAC-3’?（SEQ?ID?No.9）
Above-mentioned primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
2. extract three kinds of kinds of fish Rhabdovirus RNA respectively
1. in vial, add 600 μ l TRIZOL lysates, add the cell suspension 200 μ l that infect VHSV then, the piping and druming mixing.
2. add 200 μ l chloroforms, vibration mixing 15 sec, 4 ℃ then, 13,000 rpm, centrifugal 15 min.
3. supernatant liquor is moved in the 500 μ l Virahols, put upside down mixing, put-20 ℃ of precipitation 10 min then.
4. 4 ℃, 13,000 rpm, centrifugal 15 min.
5. inhale and abandon supernatant liquor, in vial, add 75% ethanol, 600 μ l, put upside down washing.
6. 4 ℃, centrifugal 10 min of 13,000 rpm abandon supernatant liquor.
7. 4,000 rpm, centrifugal 10 sec are thrown to the pipe bottom with the residual liquid on the tube wall, it are blotted drying at room temperature 1-5 min with micro sample adding appliance as far as possible.
8. add 100 μ l DEPC water, mixing dissolves the RNA on the tube wall gently, makes the RNA solution of VHSV.
The method of the RNA solution of preparation IHNV and SVCV is the same.
3. three kinds of viral RNA reverse transcriptions and regular-PCR amplification
(1) reverse transcription of RNA
RNA with three kinds of kinds of fish Rhabdovirus extracting is a template respectively, carries out reverse transcription with primer pdN6 immediately, and system (25 μ l) and step are as follows:
1. in the PCR pipe, add RNA template 10 μ l, pdN6 (50 μ M) 1 μ l;
2. 70 ℃, 5 min, chilling on ice;
3. continue in the PCR pipe, to add following composition, see Table 1:
Table 1
| M-MLV 5 * reaction buffer | 5.0μl |
| RNA enzyme inhibitors (40 U/ μ l) | 0.5μl |
| dNTP (10 mM) | 1.5μl |
| M-MLV (200 U/μl) | 1.0μl |
| DEPC water | 8.5μl |
4. 42 ℃, 1 h; 95 ℃, 5 min; 4 ℃, preserve.
(2) regular-PCR amplification
Be template with three kinds of RNA reverse transcription products respectively, with the corresponding primer of virus separately to carrying out pcr amplification reaction, described primer to be VP1 and VP2 primer to or IP1 and IP2 primer to or SP1 and SP2 primer right, (50 μ l) is as follows for system, sees Table 2:
Table 2
| 10× Reaction Buffer | 5.0μl |
| MgCl 2(25 mM) | 6.0μl |
| dNTP(10 mM) | 1.0μl |
| Primer 1 (25 μ M) | 1.0μl |
| Primer 2 (25 μ M) | 1.0μl |
| Taq archaeal dna polymerase (5 U/ μ l) | 1.0μl |
| Aqua sterilisa | 34μl |
| Template | 1.0μl |
Reaction conditions is as follows: 1 circulation: 95 ℃ of 5 min; 32 circulations: 95 ℃ of 45 sec, 60 ℃ of 45 sec, 72 ℃ of 1 min 45 sec; 1 circulation: 72 ℃ of 10 min; 4 ℃ of preservations.
(3) PCR product gel electrophoresis
Prepare 1.0% sepharose liquid with 1 * TAE solution, get the PCR product and carry out gel electrophoresis, observe electrophoresis result, then the PCR product of VHSV has a clear band at about 1278 bp places, the PCR product of IHNV has the PCR product of a clear band and SVCV at about 1713 bp places one clear band to be arranged at about 977 bp places, conform to expection.
4. the pulsating purifying of purpose reclaims
Respectively the PCR product of above-mentioned three kinds of viruses is carried out the purifying recovery with TakaRa DNA Fragment Purification test kit, step is as follows:
1. the DB Buffer that in 40 μ l PCR reaction solutions, adds 120 μ l, uniform mixing then.
2. the Spin Column in the test kit is placed on the Collection Tube.
3. aforesaid operations solution 1. is transferred among the Spin Column centrifugal 1 min of 12,000 rpm.
4. filtrate is added among the Spin Column more once centrifugally again, abandon filtrate then.
5. the Rinse A with 500 μ l adds among the Spin Column, and centrifugal 30 sec of 12,000 rpm abandon filtrate.
6. the Rinse B with 700 μ l adds among the Spin Column, and centrifugal 30 sec of 12,000 rpm abandon filtrate.
7. repetitive operation step 6..
8. with Spin Column by on the centrifuge tube that places 1.5 new ml, add the Elution Buffer of 60 ℃ of preheatings of 30 μ l in the centre of Spin Column film, room temperature leaves standstill 1 min.
9. the centrifugal 1 min eluted dna of 12,000 rpm.
5. pGEM-T/IN construction of recombinant plasmid
The goal gene that reclaims is connected construction recombination plasmid with pGEM-T Easy Vector, and concrete grammar and precaution are carried out to specifications.(10 μ l) is as follows for linked system, table 3:
Table 3
| 10 * ligation buffer(connects damping fluid | 1.0 μl |
| pGEM-T Easy Vector | 1.0 μl |
| PCR reclaims product | 2.0 μl |
| T4 DNA ligase enzyme | 1.0 μl |
| Aqua sterilisa | 5.0 μl |
Abundant mixing, low speed is instantaneous centrifugal, and after room temperature was placed 2 h, 16 ℃ were spent the night.
6. transform
To connect product and transform DH5 α competent cell, concrete steps are as follows:
1. from-80 ℃ of taking-up DH5 α competent cells, room temperature places on the frozen water after dissolving rapidly at once.
2. get 100 μ l competent cells and be added in the 10 μ l connection mixture, gently the pressure-vaccum mixing.
3. leave standstill in the ice-water bath about 30 min.
4. 42 ℃ of water-bath thermal shock 90 sec are put in cooled on ice 2-5 min at once.
5. add 900 μ l SOC, place 37 ℃ of temperature to bathe 60 min.
6. evenly smear 40 μ l IPTG and 40 μ l X-gal stock solutions at the LB planar surface that contains penbritin (100 μ g/ml), place 37 ℃ of incubators.
7. with the competent cell of temperature bath, centrifugal 3 min of 5000 rpm remove supernatant, stay about 100 μ l liquid suspension bacteriums, evenly are applied on the LB/Amp/IPTG/X-gal flat board.
8. 37 ℃ of incubators are cultivated 12-16 h.
The evaluation of 7 plasmids
(1) extraction of plasmid
Extract plasmid with TaKaRa MiniBEST Plasmid Purification Kit Ver 2.0 test kits, concrete operations are as follows:
1. colibacillary cultivation: select white single colony inoculation to the LB/AMP liquid nutrient medium of about 5 ml from plate culture medium, 37 ℃, cultivate 8-12 h.
2. get the incubated overnight bacterium liquid of 1-4 ml, centrifugal 2 min of 12,000 rpm abandon supernatant.
3. bacterial precipitation fully suspends to use the Solution I (containing RNase A1) of 250 μ l.
4. the Solution II that adds 250 μ l spins upside down lightly mixes 5-6 time, makes the abundant cracking of thalline, forms clear solution.
5. the Solution III that adds 4 ℃ of precoolings of 400 μ l spins upside down gently and mixes 5-6 time, and until forming consolidation aggegation piece, room temperature leaves standstill 2 min then.Centrifugal 10 min of 12,000 rpm get supernatant then.
6. the Spin Column in the test kit is placed on the Collection Tube, the supernatant liquor of aforesaid operations in 5. is transferred among the Spin Column, centrifugal 1 min of 12,000 rpm abandons filtrate.
7. the Rinse A with 500 μ l adds among the Spin Column, and centrifugal 30 sec of 12,000 rpm abandon filtrate.
8. the Rinse B with 700 μ l adds among the Spin Column, and centrifugal 30 sec of 12,000 rpm abandon filtrate.
9. repetitive operation step 8..
10. Spin Column is placed on the centrifuge tube of 1.5 new ml, adds the Elution Buffer of 60 ℃ of preheatings of 60 μ l in the centre of Spin Column film, room temperature leaves standstill 1 min, then the centrifugal 1 min eluted dna of 12,000 rpm.
(2) enzyme of plasmid is cut evaluation
Plasmid with extracting carries out enzyme with EcoR I and cuts evaluation, 37 ℃ of reaction 2 h, and 1.0% agarose gel electrophoresis is observed enzyme and is cut the result.Then the recombinant plasmid enzyme of VHSV is cut product has a clear band at about 1278 bp places, and the recombinant plasmid enzyme of IHNV is cut product to be had the recombinant plasmid enzyme of a clear band and SVCV to cut product at about 1713 bp places one clear band to be arranged, conform to expection at about 977 bp places.
(3) order-checking is identified
Enzyme is cut the single positive colony order-checking (worker is given birth in Shanghai) of evaluation.Sequencing result is: the N gene corresponding DNA sequences of VHSV is the nucleotide sequence shown in the sequence table SEQ ID No.1; The N gene corresponding DNA sequences of IHNV is the nucleotide sequence shown in the sequence table SEQ ID No.2; The N gene corresponding DNA sequences of SVCV is the nucleotide sequence shown in the sequence table SEQ ID No.3.
8. the N gene in vitro of three kinds of kinds of fish Rhabdovirus is transcribed
(1) processing of template
1. with the Mlu I plasmid is carried out enzyme and cut, (20 μ l) is as follows for reaction system, table 4:
Table 4
| 10×H Buffer | 2.0μl |
| Mlu I | 1.0μl |
| Dna profiling | ≤1 μg |
| Aqua sterilisa | To 20 μ l |
2. 37 ℃ are reacted 8 h;
3. 1.0% agarose gel electrophoresis is observed enzyme and is cut the result, and after Mlu I enzyme was cut, the recombinant plasmid of VHSV had a clear band at about 4293 bp places; The recombinant plasmid of IHNV has a clear band at about 3992 bp places, and the recombinant plasmid of SVCV has a clear band at about 4728 bp places, conforms to expection;
4. with TakaRa DNA Fragment Purification test kit enzyme is cut product and carry out the purifying recovery, the concrete operations step is with 4.
(2) in-vitro transcription
1. cut the linear segment that obtains as template with Mlu I enzyme, carry out in-vitro transcription with Promega RiboMAX Large Scale RNA Production System-T7 test kit, (50 μ l) is as follows for reaction system, table 5:
Table 5
| T7 Transcription 5X Buffer | 10μl |
| rNTPs ( 25 mM ATP, CTP, GTP, UTP ) | 15μl |
| Linear dna profiling (5-10 μ g total) and DEPC treating water | 20μl |
| Enzyme Mix (T7) | 5.0μl |
2. mixing is put 37 ℃, and incubation 4 h add 1 μ l RQ1 DNA enzyme then in reaction system, and 37 ℃ of incubation 30 min remove dna profiling;
3. extract purifying with TRIZOL, concrete operations precipitate with 200 μ l DEPC water dissolution RNA at last with step 2.
9. Determination on content and the dilution of three kinds of kinds of fish Rhabdovirus in-vitro transcription RNA
With the RNA fragment of the VHSV in-vitro transcription behind the purifying, carry out 100 times of dilutions, be 0.087 with the spectrophotometer OD value that to record its OD value at 260 nm wavelength places be 0.176,280 nm wavelength place then.OD
260/OD
280=2.02。Calculating RNA concentration is 9.19 * 10
11Copy/μ l is diluted to 10 with sample with 75% ethanolic soln
9Copy/μ l, and sample carried out 10 times of serial gradient dilutions, obtain concentration and be respectively 10
9, 10
8, 10
7, 10
6, 10
5, 10
4, 10
3, 10
2, the RNA solution of 10,1.0 copy/μ l.
With the RNA fragment of the IHNV in-vitro transcription behind the purifying, carry out 10 times of dilutions, be 0.362 with the spectrophotometer OD value that to record its OD value at 260 nm wavelength places be 0.655,280 nm wavelength place then.OD
260/OD
280=1.8。Calculating RNA concentration is 4.36 * 10
11Copy/μ l.Sample is diluted to 10 with 75% ethanolic soln
9Copy/μ l, and sample carried out 10 times of serial dilutions, obtain concentration and be respectively 10
9, 10
8, 10
7, 10
6, 10
5, 10
4, 10
3, 10
2, the RNA solution of 10,1.0 copy/μ l.
With the RNA fragment of the SVCV in-vitro transcription behind the purifying, carry out 10 times of dilutions, be 0.063 with the spectrophotometer OD value that to record its OD value at 260 nm wavelength places be 0.126,280 nm wavelength place then.OD
260/OD
280=2.0。Calculating RNA concentration is 2.4 * 10
10Copy/μ l.Sample is diluted to 10 with 75% ethanolic soln
9Copy/μ l, and sample carried out 10 times of serial dilutions, obtain concentration and be respectively 10
9, 10
8, 10
7, 10
6, 10
5, 10
4, 10
3, 10
2, the RNA solution of 10,1.0 copy/μ l.
Above-mentioned 75% ethanolic soln is prepared with DEPC water, described DEPC water: twice distillation of tap water, and through Millipore MILLI-Q PF PLUS pure water instrument purifying, resistivity 〉=18.0M Ω .cm.
10. the copy number of three kinds of kinds of fish Rhabdovirus measurement audit samples is quantitative
Use in order to be fit to measure audit, increase the concentration of the RNA solution of above-mentioned three kinds of kinds of fish Rhabdovirus, and use the copy number that the fluorescence RT-PCR method is come each concentration of indirect measurement RNA solution, the result is as follows:
Primer and probe can be selected sequence well known in the prior art in the fluorescence RT-PCR method, and cycling condition is set to:
Fs, 42 ℃ of 30 min of reverse transcription;
Subordinate phase, pre-92 ℃ of 3 min of sex change;
Phase III, 92 ℃ of 10 s; 45 ℃ of 30 s; 72 ℃ of 1 min; 5 circulations;
The quadravalence section, 92 ℃ of 10 s; 60 ℃ of 30 s; Fluorescence is collected in 40 circulations when the each round-robin annealing of quadravalence section is extended.
（1）VHSV
Sample 1: contain purpose nucleic acid fragment 10
975% the ethanolic soln of copy/mL, all positive through 10 test result of samples of picked at random, Ct mean value is 10.16.
Sample 2: contain purpose nucleic acid fragment 10
875% the ethanolic soln of copy/mL, all positive through 10 test result of samples of picked at random, Ct mean value is 13.84.
Sample 3: contain purpose nucleic acid fragment 10
775% the ethanolic soln of copy/mL, all positive through 10 test result of samples of picked at random, Ct mean value is 17.54.
Sample 4: do not contain 75% ethanolic soln of purpose nucleic acid fragment, all negative through 10 test result of samples of picked at random, Ct〉40.
（2）IHNV
Sample 1: contain purpose nucleic acid fragment 10
975% the ethanolic soln of copy/mL, all positive through 10 test result of samples of picked at random, Ct mean value is 9.48.
Sample 2: contain purpose nucleic acid fragment 10
875% the ethanolic soln of copy/mL, all positive through 10 test result of samples of picked at random, Ct mean value is 12.89.
Sample 3: contain purpose nucleic acid fragment 10
775% the ethanolic soln of copy/mL, all positive through 10 test result of samples of picked at random, Ct mean value is 16.30.
Sample 4: do not contain 75% ethanolic soln of purpose nucleic acid fragment, all negative through 10 test result of samples of picked at random, Ct〉40.
（3）SVCV
Sample 1: contain purpose nucleic acid fragment 10
975% the ethanolic soln of copy/mL, all positive through 10 test result of samples of picked at random, Ct mean value is 10.69.
Sample 2: contain purpose nucleic acid fragment 10
875% the ethanolic soln of copy/mL, all positive through 10 test result of samples of picked at random, Ct mean value is 15.17.
Sample 3: contain purpose nucleic acid fragment 10
775% the ethanolic soln of copy/mL, all positive through 10 test result of samples of picked at random, Ct mean value is 18.23.
Sample 4: do not contain 75% ethanolic soln of purpose nucleic acid fragment, all negative through 10 test result of samples of picked at random, Ct〉40.
To sum up, according to measurement result, the final sample 2 of determining to select three kinds of viruses, sample 3 and sample 4 are as measuring the audit sample.And determine 10
7Copy/mL purpose nucleic acid fragment solution, its relative reference Ct value about 17,10
8Copy/mL purpose nucleic acid fragment solution, its relative reference Ct value is about 13, adopts two sample mean test of significance-t check carrying out statistical study.
Embodiment 2 measures the composition of audit with test kit
Form test kit with the above-mentioned quantitative RNA solution of copy number that carries out as positive, be specially:
A: contain 10
775% ethanolic soln of copy/mL purpose nucleic acid fragment, described purpose nucleic acid fragment are the RNA fragment of viral haemorrhagic septicaemia virus (VHSV) in-vitro transcription, and its corresponding DNA sequences is the nucleotide sequence shown in the sequence table SEQ ID No.1;
B: contain 10
875% ethanolic soln of copy/mL purpose nucleic acid fragment, described purpose nucleic acid fragment are the RNA fragment of viral haemorrhagic septicaemia virus (VHSV) in-vitro transcription, and its corresponding DNA sequences is the nucleotide sequence shown in the sequence table SEQ ID No.1;
C: contain 10
775% ethanolic soln of copy/mL purpose nucleic acid fragment, described purpose nucleic acid fragment are the RNA fragment of the sick virus of infectivity hematopoietic tissue necrosis (IHNV) in-vitro transcription, and its corresponding DNA sequences is the nucleotide sequence shown in the sequence table SEQ ID No.2;
D: contain 10
875% ethanolic soln of copy/mL purpose nucleic acid fragment, described purpose nucleic acid fragment are the RNA fragment of the sick virus of infectivity hematopoietic tissue necrosis (IHNV) in-vitro transcription, and its corresponding DNA sequences is the nucleotide sequence shown in the sequence table SEQ ID No.2;
E: contain 10
775% ethanolic soln of copy/mL purpose nucleic acid fragment, described purpose nucleic acid fragment are the RNA fragment of SVCV (SVCV) in-vitro transcription, and its corresponding DNA sequences is the nucleotide sequence shown in the sequence table SEQ ID No.3;
F: contain 10
875% ethanolic soln of copy/mL purpose nucleic acid fragment, described purpose nucleic acid fragment are the RNA fragment of SVCV (SVCV) in-vitro transcription, and its corresponding DNA sequences is the nucleotide sequence shown in the sequence table SEQ ID No.3;
G: the solution that does not contain the purpose nucleic acid fragment.
Embodiment 3 measures the use of audit with test kit
1. the world, country and industry standard method or other nonstandard methods can be selected in each laboratory of participating in the experiment, and carry out the detection validation of three kinds of kinds of fish Rhabdovirus nucleic acid;
2. test kit specification sheets
2.1 storage temperature :-20 ℃;
2.2 validity period: 6 months;
2.3 using method: put upside down the mixing liquid in pipe before the use, and then extracting RNA nucleic acid carries out test analysis.
2.4 cycling condition setting:
Fs, 42 ℃ of 30 min of reverse transcription;
Subordinate phase, pre-92 ℃ of 3 min of sex change;
Phase III, 92 ℃ of 10 s; 45 ℃ of 30 s; 72 ℃ of 1 min; 5 circulations;
The quadravalence section, 92 ℃ of 10 s; 60 ℃ of 30 s; Fluorescence is collected in 40 circulations when the each round-robin annealing of quadravalence section is extended.
After test detects and finishes, according to the fluorescence curve and the Ct value result of determination of collecting.
2.5 relative reference Ct value: be as the criterion 10 with the loop parameter in 2.4
7The relative reference Ct value of copy/mL purpose nucleic acid fragment solution is about 17,10
8The relative reference Ct value of copy/mL purpose nucleic acid fragment solution is about 13.
3. evaluation of result
If do not detect the specific RNA sequence in this test kit, illustrate that then this laboratory detection technology of participating in the experiment has problem; If can detect the specific purpose nucleic acid fragment in this test kit, and the Ct value that detects and the Ct value employing two sample mean test of significance-t that demarcates check and carry out statistical study and do not have significant difference, proves that then this laboratory detection technology level of participating in the experiment is good; If can detect the specific purpose nucleic acid fragment in this test kit, two sample mean test of significance-statistical study is carried out in the t check, and there were significant differences but the Ct value that detects adopts with the Ct value of demarcating, then prove this not enough standard of laboratory detection technology level and stable of participating in the experiment, need to improve.
Embodiment 4 measures requirement and homogeneity and the stability test of audit with each sample of test kit
1. sample packing: embodiment 1 preparation and the accurate packing of sample extraction of demarcating, tolerance range should be in ± 1%.
2. the cooperation of tiring is demarcated: invite outside laboratory fellowship that the copy number of sample is demarcated.After the sample process long-distance transport with the ice bag preservation, the outside laboratory of invitation is tested sample, and the Ct value of its detection is consistent with the characteristic value of sample, has proved the accuracy of this sample characteristics of for example value test.
3. the uncertainty of sample characteristics of for example value is explored
By analysis, determine the also source of simplified measurement uncertainty to fluorescence quantitative PCR detection DNA measuring process; Adopt type A evaluation of uncertainty method and uncertainty type B evaluation method, quantize each uncertainty component; Determine synthetic uncertainty and expanded uncertainty.As a result, uncertainty of measurement source mainly contains: repeatability in batch, batch between factors such as repeatability and method bias.The expanded uncertainty U=0.62 of quantitative fluorescent PCR mensuration DNA (k=1.96, n=2); In each uncertainty component, the uncertainty that is caused by the method bias accounts for the largest percentage.(quantitative fluorescent PCR is measured uncertainty evaluation and the application of HBV DNA, Chinese laboratory medicine magazine, 2007 2 phases.）
4. the uniformity testing of sample
Randomly draw 10 duplicate samples, carry out fluorescence RT-PCR and detect, calculate the variation coefficient according to the Ct value that records, pipe differences≤5%(is the variation coefficient≤5%).
Sample A uniformity test result in table 6 test kit
| Sample number into spectrum | The 1st test CT value | Detect the CT value the 2nd time | Mean value |
| A-1 | 17.64 | 17.53 | 17.59 |
| A -2 | 17.44 | 17.32 | 17.38 |
| A -3 | 17.66 | 17.75 | 17.71 |
| A -4 | 18.20 | 17.98 | 18.09 |
| A -5 | 17.49 | 18.01 | 17.75 |
| A -6 | 17.24 | 17.09 | 17.17 |
| A -7 | 17.53 | 17.56 | 17.55 |
| A -8 | 18.20 | 17.89 | 18.05 |
| A -9 | 17.88 | 17.72 | 17.80 |
| A -10 | 16.99 | 17.05 | 17.02 |
| Mean value | 17.63 | 17.59 | 17.61 |
Sample B uniformity test result in table 7 test kit
| Sample number into spectrum | The 1st test CT value | Detect the CT value the 2nd time | Mean value |
| B -1 | 12.23 | 12.33 | 12.28 |
| B -2 | 12.61 | 12.46 | 12.54 |
| B -3 | 13.22 | 13.18 | 13.20 |
| B -4 | 12.19 | 12.87 | 12.53 |
| B-5 | 13.56 | 13.29 | 13.43 |
| B-6 | 13.33 | 13.67 | 13.50 |
| B -7 | 12.88 | 13.70 | 13.29 |
| B -8 | 13.66 | 13.26 | 13.43 |
| B -9 | 13.64 | 12.93 | 13.29 |
| B -10 | 13.31 | 13.57 | 13.44 |
| Mean value | 13.06 | 13.13 | 13.10 |
Sample C uniformity test result in table 8 test kit
| Sample number into spectrum | The 1st test CT value | Detect the CT value the 2nd time | Mean value |
| C-1 | 16.30 | 16.53 | 16.42 |
| C -2 | 16.44 | 17.01 | 16.73 |
| C -3 | 16.66 | 17.15 | 16.91 |
| C -4 | 16.20 | 16.91 | 16.56 |
| C -5 | 17.49 | 16.36 | 16.93 |
| C -6 | 17.24 | 16.09 | 16.67 |
| C -7 | 16.53 | 16.88 | 16.71 |
| C -8 | 16.20 | 16.56 | 16.38 |
| C -9 | 16.88 | 16.70 | 16.79 |
| C -10 | 15.99 | 16.75 | 16.39 |
| Mean value | 16.59 | 16.69 | 16.64 |
Sample D uniformity test result in table 9 test kit
| Sample number into spectrum | The 1st test CT value | Detect the CT value the 2nd time | Mean value |
| D-1 | 12.89 | 12.87 | 12.88 |
| D -2 | 12.71 | 12.46 | 12.59 |
| D -3 | 13.22 | 12.18 | 12.70 |
| D -4 | 12.29 | 12.37 | 12.33 |
| D -5 | 13.06 | 12.29 | 12.68 |
| D -6 | 13.33 | 13.17 | 13.25 |
| D -7 | 12.88 | 12.77 | 12.83 |
| D -8 | 13.26 | 12.26 | 12.76 |
| D -9 | 12.64 | 11.93 | 12.29 |
| D -10 | 11.98 | 12.57 | 12.28 |
| Mean value | 12.83 | 12.49 | 12.66 |
Sample E uniformity test result in table 10 test kit
| Sample number into spectrum | The 1st test CT value | Detect the CT value the 2nd time | Mean value |
| E-1 | 18.62 | 18.53 | 18.58 |
| E -2 | 18.43 | 17.72 | 18.08 |
| E -3 | 18.46 | 18.32 | 18.39 |
| E -4 | 18.86 | 18.98 | 18.92 |
| E -5 | 18.69 | 18.01 | 18.35 |
| E -6 | 18.24 | 17.09 | 17.67 |
| E -7 | 18.53 | 17.56 | 18.05 |
| E -8 | 19.22 | 17.83 | 18.53 |
| E -9 | 19.23 | 18.75 | 18.99 |
| E -10 | 17.90 | 17.05 | 17.48 |
| Mean value | 18.62 | 17.98 | 18.30 |
Sample F uniformity test result in table 11 test kit
| Sample number into spectrum | The 1st test CT value | Detect the CT value the 2nd time | Mean value |
| F-1 | 14.23 | 15.18 | 14.71 |
| F-2 | 14.61 | 14.46 | 14.54 |
| F -3 | 15.33 | 14.33 | 14.83 |
| F -4 | 14.19 | 14.87 | 14.53 |
| F -5 | 15.56 | 14.93 | 15.25 |
| F -6 | 15.64 | 15.37 | 15.51 |
| F -7 | 14.88 | 15.20 | 15.04 |
| F -8 | 15.66 | 15.26 | 15.46 |
| F -9 | 15.22 | 15.09 | 15.16 |
| F -10 | 15.31 | 15.52 | 15.42 |
| Mean value | 15.06 | 15.02 | 15.04 |
Sample G uniformity test result in table 12 test kit
| Sample number into spectrum | The 1st test CT value | Detect the CT value the 2nd time | Mean value |
| G-1 | >40 | >40 | >40 |
| G -2 | >40 | >40 | >40 |
| G -3 | >40 | >40 | >40 |
| G -4 | >40 | >40 | >40 |
| G -5 | >40 | >40 | >40 |
| G -6 | >40 | >40 | >40 |
| G -7 | >40 | >40 | >40 |
| G -8 | >40 | >40 | >40 |
| G -9 | >40 | >40 | >40 |
| G -10 | >40 | >40 | >40 |
| Mean value | >40 | >40 | >40 |
5. stability of sample check
Extract the standard substance of preparation at random respectively, under following every kind of condition, place 10: 20 ℃～25 ℃ of (1) room temperatures, relative humidity 20%～50%, 14d takes out and tests; (2) the refrigerator cold-storage temperature is 2 ℃～8 ℃, and taking-up in 6 months is tested; (3)-20 ℃, take out after 1 year and test; (4) contrast ,-80 ℃, the long-term placement.Extract RNA respectively.Use the fluorescence RT-PCR method and come the copy number of the above-mentioned RNA of indirect measurement.Adopt two sample mean test of significance-t check carrying out statistical study.As a result, place under above-mentioned each condition, sample stability is all fine.
Sequence table
＜110〉People's Republic of China Beijing Entry-Exit Inspection and Quarantine Bureau
＜120〉the measurement audit test kit of three kinds of kinds of fish Rhabdovirus of a kind of detection
<130>
<160> 9
<170> PatentIn?version?3.3
<210> 1
<211> 1278
<212> DNA
＜213〉the N gene of VHSV
<400> 1
atggaaggag?gaattcgtgc?agcgttttca?ggcctgaatg?atgttaggat?cgaccccacc 60
ggtggagagg?gacgggtact?tgtacctggt?gaagtggaac?tcatcgtgta?tgttggtgga 120
tttggtgagg?aagataggac?ggtgattgtg?gatgcactct?ccgcactcgg?gggaccccag 180
actgtgcagg?cgttgtccgt?gcttctctcc?tatgtactcc?aagggaatac?acaggaggac 240
ctagaaacaa?agtgcaaggt?cctcacagac?atgggcttca?aggtgacaca?ggcagccagg 300
gctacgagca?tcgaggcagg?aatcatgatg?cccatgagag?aactggccct?gactgtcaat 360
gacgacaacc?tcatggaaat?cgtgaagggg?accttgatga?catgctccct?tctgaccaag 420
tactcggtgg?acaagatgat?caagtacatc?accaagaaac?tcggggagct?ggcagacact 480
cagggagttg?gggaactgca?gcacttcacc?gctgacaagg?cagccatcag?gaaactcgca 540
gggtgtgtgc?gtcccgggca?gaagatcacc?aaggccctct?atgcattcat?cctgaccgag 600
atcgcagacc?ccaccaccca?gtcgagggcc?cgagccatgg?gggcgttgag?gctcaacggg 660
acaggaatga?ccatgattgg?gctgttcacc?caggccgcca?acaacttggg?cattgccccg 720
gcaaagctgc?tggaggacct?ctgcatggag?tccctggttg?agtcagccag?acggatcatc 780
cagttgatga?gacaggtgtc?agaggcgaag?tccatccaag?agcgctacgc?catcatgatg 840
agtcggatgc?tgggagagtc?ctactacaag?tcgtatggac?tcaatgacaa?ctccaagatc 900
tcttacattc?tgtcacagat?cagtgggaag?tacgcagtgg?actccctgga?aggcctggag 960
gggatcaagg?tgacagagaa?gttccgcgag?ttcgctgagc?ttgttgcaga?agtcctggtg 1020
gacaagtacg?agaggattgg?agaggacagc?acggaggtct?cagatgtcat?cagggaggcg 1080
gccagacagc?acgcgcgcag?gacatccgcc?aagccagagc?caaaggcccg?caacttcagg 1140
agctccaccg?gaagggggaa?ggagcaggag?acgggggggt?ccgatgatga?cgactacccc 1200
gaggactctg?actaaaagac?actcccgtct?cataaccaac?atagatagaa?aaaaacggca 1260
cgattataag?aatttctc 1278
<210> 2
<211> 977
<212> DNA
＜213〉the N gene of IHNV
<400> 2
tcacgaacga?tgacaagcgc?actcagagag?acgttcactg?gactcagaga?catcaagggg 60
ggagtcctcg?aggatgcaga?gacggagtat?cgtcccggta?cgataaccct?ccctctctat 120
ttctccaagg?cagactttga?cctagagatg?atcaagcggg?cggtgagtca?cgtcggagga 180
gagggaacga?gaagggcatt?gggcctcctg?tgcgcgttcg?tcattgcaga?gacggtccca 240
tcggggacag?gcacggccgc?cgaacctctg?gaagccctgg?gcttcttgct?ggagtctttg 300
gacactgggg?caccactgga?cgttaccttc?gcagatccca?acaacaagct?tgcagaaacg 360
atctcaaagg?aaaatgtcct?tgaggttgtg?accggcctcc?tcttcacctg?cgccctactg 420
acaaagtatg?atgtggacaa?gatggccaca?tactgccaaa?acaagctcga?gcgtcttgca 480
accagccaag?ggattgacga?gttggtcaac?ttcaacgcca?acaggggagt?gctggccaag 540
atcggggcgg?tacttaggcc?tggacagaag?ctcaccaagg?ctatctatgg?gatcattctc 600
atcaacctgt?ccgacccagc?caccgccgct?agagccaagg?cactgtgcgc?catgagactg 660
agcgggacgg?gaatgacaat?ggtgggactg?ttcaaccaag?ccgcaaagaa?cctgggcgcc 720
cttccagccg?accttctaga?agatctgtgc?atgaagtcag?tggtggagtc?cgccagacgc 780
attgtcagac?tgatgaggat?cgtagcagag?gccccagggg?tagcagcaaa?gtacggcgtc 840
atgatgagca?gatgctcggg?ggaggggtac?ttcaaggcct?acgggatcaa?cgagaacgcc 900
aggatcacct?gcattctcat?gaacatcaac?gataggtatg?acgaggggac?ctcgagagga 960
ctgacaggga?taaaggt 977
<210> 3
<211> 1713
<212> DNA
＜213〉the N gene of SVCV
<400> 3
gttccttccc?tgttcatggg?agattaagtt?gagatcaata?ttcgctgaga?tatgaaaaaa 60
actaacagac?atcatgtcta?tcatcagcta?catcgcattc?cttttgctaa?ttgattccac 120
attgggaatc?cccatatttg?ttccatccgg?gcggaatata?tcagggcaac?ctgtaattca 180
gccatttgat?tatcaatgtc?caatacacgg?aaatctacct?aacacaatgg?gattgagtgc 240
caccaaattg?acaataaaat?ctccatctgt?cttcagtaca?gataaagttt?ctggatggat 300
ctgccatgca?gctgaatgga?aaacaacttg?tgattacaga?tggtacggac?cccaatatat 360
aacccacagt?attcatccaa?tcagtcctac?catagatgaa?tgcaagagaa?tcatttcaag 420
gattgcatca?ggaactgatg?aagatctggg?gtttccccct?caaagttgcg?gatgggcatc 480
tgtcacaaca?gtgtcaaata?ctaattacaa?ggtagtaccc?cattccgttc?atttggggcc 540
gtacggagga?cactggatcg?atcatgaatt?caatgggggc?gaatgcagag?aaaaagtgtg 600
tgaaatgaaa?gggaaccact?ctatttggat?cacagatgag?accgtgcagc?atgaatgtga 660
aaagcacata?gaggaagttg?aaggaattat?gtacgggaat?gctccgagag?gggatgcaat 720
atatattaac?aactttatta?tagataaaca?tcatagagta?tacagattcg?gggggtcttg 780
tcgaatgaaa?ttctgtaata?aagatggtat?aaaattcaca?agaggagact?gggtagaaaa 840
aacagctgga?acattgacga?atatttatgc?aaatatacct?gaatgtgctg?atggaacgtt 900
ggtatctggt?caccgacctg?gattagactt?gattgacaca?gtcttcaatt?tggaaaatgt 960
ggtagaatat?actttgtgtg?aagggactaa?aagaaaaatc?aataaccaag?aaaagttgac 1020
gtcagtggat?ttgagttatt?tggccccaag?aattggaggg?tttggatcag?tattcagagt 1080
gagaaacgga?acattagaga?gagggagcac?tacttatatc?aagatagaag?tagagggacc 1140
tattgtcgac?tcgttgaatg?gaacagatcc?gagaaccaac?gcctcaagag?tattttggga 1200
cgactgggag?ttagatggca?atatatatca?gggctttaat?ggtgtatata?aagggaaaga 1260
tgggaagatc?catattccct?tgaatatgat?agaatcagga?atcatagatg?atgaacttcg 1320
acatgctttc?caagccgata?ttatccctca?tcctcattat?gacgacgatg?aaatccgaga 1380
ggacgatata?ttcttcgata?atactggaga?aaatggaaat?cccgtggatg?cagtggtaga 1440
atgggtcagt?gggtggggaa?ctagtctaaa?attctttggc?acgactctgg?tcgccctgat 1500
tttgatcttt?ctgctcatca?ggtgctgtgt?tgcttgcact?tatttgatga?agaagagtaa 1560
acggcctgca?acagaatcac?acgaaatgcg?gtccttcgtt?tgagagatag?ccaattttaa 1620
gcaaagacca?agatattatc?ttaataggtg?tatgaaaaaa?actataacag?acatcatgtt 1680
tgagtgggaa?agtcaagata?ctccgtctgg?att 1713
<210> 4
<211> 22
<212> DNA
＜213〉synthetic primer VP1
<400> 4
atggaaggag?gaattcgtgc?ag 22
<210> 5
<211> 23
<212> DNA
＜213〉synthetic primer VP2
<400> 5
gagaaattct?tataatcgtg?ccg 23
<210> 6
<211> 24
<212> DNA
＜213〉synthetic primer I P1
<400> 6
tcacgaacga?tgacaagcgc?actc 24
<210> 7
<211> 24
<212> DNA
＜213〉synthetic primer I P2
<400> 7
acctttatcc?ctgtcagtcc?tctc 24
<210> 8
<211> 22
<212> DNA
＜213〉synthetic primer SP1
<400> 8
gttccttccc?tgttcatggg?ag 22
<210> 9
<211> 22
<212> DNA
＜213〉synthetic primer SP2
<400> 9
aatccagacg?gagtatcttg?ac 22
Claims (2)
1. test kit is used in a measurement audit that detects three kinds of kinds of fish Rhabdovirus, it is characterized in that this test kit is made up of following sample:
A: contain 10
7The solution of copy/mL purpose nucleic acid fragment, the RNA fragment that described purpose nucleic acid fragment is the viral haemorrhagic septicaemia virus in-vitro transcription, its corresponding DNA sequences are the nucleotide sequence shown in the sequence table SEQ ID No.1;
B: contain 10
8The solution of copy/mL purpose nucleic acid fragment, the RNA fragment that described purpose nucleic acid fragment is the viral haemorrhagic septicaemia virus in-vitro transcription, its corresponding DNA sequences are the nucleotide sequence shown in the sequence table SEQ ID No.1;
C: contain 10
7The solution of copy/mL purpose nucleic acid fragment, described purpose nucleic acid fragment are the RNA fragment of the sick viral in-vitro transcription of infectivity hematopoietic tissue necrosis, and its corresponding DNA sequences is the nucleotide sequence shown in the sequence table SEQ ID No.2;
D: contain 10
8The solution of copy/mL purpose nucleic acid fragment, described purpose nucleic acid fragment are the RNA fragment of the sick viral in-vitro transcription of infectivity hematopoietic tissue necrosis, and its corresponding DNA sequences is the nucleotide sequence shown in the sequence table SEQ ID No.2;
E: contain 10
7The solution of copy/mL purpose nucleic acid fragment, the RNA fragment that described purpose nucleic acid fragment is the SVCV in-vitro transcription, its corresponding DNA sequences are the nucleotide sequence shown in the sequence table SEQ ID No.3;
F: contain 10
8The solution of copy/mL purpose nucleic acid fragment, the RNA fragment that described purpose nucleic acid fragment is the SVCV in-vitro transcription, its corresponding DNA sequences are the nucleotide sequence shown in the sequence table SEQ ID No.3;
G: the solution that does not contain the purpose nucleic acid fragment.
2. test kit according to claim 1 is characterized in that, described solution is 75% ethanolic soln.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876810A (en) * | 2012-10-19 | 2013-01-16 | 中国水产科学研究院长江水产研究所 | RT-PCR (reverse transcription-polymerase chain reaction) detection kit and detection method for spring viraemia of carp virus |
| CN103173574A (en) * | 2013-04-03 | 2013-06-26 | 山东出入境检验检疫局检验检疫技术中心 | Method for detecting spring viremia of carp virus based on liquid chip |
| CN105002298A (en) * | 2015-05-21 | 2015-10-28 | 上海市水产研究所 | Fluorescent quantitative PCR detection method of spring viraemia of carp virus |
| CN105112566A (en) * | 2015-09-14 | 2015-12-02 | 山东出入境检验检疫局检验检疫技术中心 | Infectious hematopoietic necrosis virus detection kit based on pyrosequencing |
| CN110592272A (en) * | 2018-06-13 | 2019-12-20 | 杭州众测生物科技有限公司 | RAA constant temperature fluorescence detection method and reagent for Viral Hemorrhagic Septicemia (VHSV) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101440411A (en) * | 2008-12-17 | 2009-05-27 | 中华人民共和国北京出入境检验检疫局 | Reagent kit and nucleotide sequence for detecting three kinds of fish Rhabdovirus |
-
2011
- 2011-04-27 CN CN2011101065461A patent/CN102212620A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101440411A (en) * | 2008-12-17 | 2009-05-27 | 中华人民共和国北京出入境检验检疫局 | Reagent kit and nucleotide sequence for detecting three kinds of fish Rhabdovirus |
Non-Patent Citations (2)
| Title |
|---|
| 许建明: "Taqman MGB探针快速定量检测VHSV方法的研究", 《高技术通讯》, vol. 20, no. 2, 31 December 2010 (2010-12-31), pages 208 - 213 * |
| 许建明: "三重实时荧光RT-PCR检测三种鱼类弹状病毒的研究", 《检验检疫学刊》, vol. 19, no. 1, 31 December 2009 (2009-12-31), pages 7 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876810A (en) * | 2012-10-19 | 2013-01-16 | 中国水产科学研究院长江水产研究所 | RT-PCR (reverse transcription-polymerase chain reaction) detection kit and detection method for spring viraemia of carp virus |
| CN103173574A (en) * | 2013-04-03 | 2013-06-26 | 山东出入境检验检疫局检验检疫技术中心 | Method for detecting spring viremia of carp virus based on liquid chip |
| CN105002298A (en) * | 2015-05-21 | 2015-10-28 | 上海市水产研究所 | Fluorescent quantitative PCR detection method of spring viraemia of carp virus |
| CN105002298B (en) * | 2015-05-21 | 2018-08-31 | 上海市水产研究所 | A kind of fluorescent quantitative PCR detection method of huichun viremia virus |
| CN105112566A (en) * | 2015-09-14 | 2015-12-02 | 山东出入境检验检疫局检验检疫技术中心 | Infectious hematopoietic necrosis virus detection kit based on pyrosequencing |
| CN110592272A (en) * | 2018-06-13 | 2019-12-20 | 杭州众测生物科技有限公司 | RAA constant temperature fluorescence detection method and reagent for Viral Hemorrhagic Septicemia (VHSV) |
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